Zebrafish etv2 knock‐in line labels vascular endothelial and blood progenitor cells

Abstract

Background: ETS transcription factor Etv2/Etsrp is one of the earliest markers for vascular and hematopoietic progenitors and functions as a key regulator of hematovascular development in multiple vertebrates, including zebrafish. Therefore, transgenic etv2 reporter lines provide a valuable tool to study vasculogenesis and hematopoiesis. However, previously generated zebrafish reporter lines do not fully recapitulate the endogenous pattern of etv2 expression. Results: Here we used CRISPR/Cas9-mediated homology-independent DNA repair approach to knock-in a Gal4 transcriptional activator into the zebrafish etv2 genomic locus, thus generating etv2 ci32Gt gene trap line. etv2 ci32Gt ; UAS:GFP embryos show GFP expression in vascular endothelial, myeloid and red blood cells. Because gal4 insertion interrupts the etv2 locus, homozygous etv2 ci32Gt embryos display defects in vasculogenesis and myelopoiesis, and enable visualizing etv2-deficient hematovascular progenitors in live embryos. Furthermore, we performed differential transcriptome analysis of sorted GFPpositive cells from heterozygous and homozygous etv2 ci32Gt embryos. Approximately 500 downregulated genes were identified in etv2 ci32Gt homozygous embryos, which include multiple genes expressed in vascular endothelial and myeloid cells. Conclusions: The etv2 ci32Gt gene trap line and the data sets of misregulated genes will be valuable resources to study hematopoietic and vascular development. K E Y W O R D SCas9, CRISPR, myeloid, red blood cell, RNA-seq, transcriptome, vascular endothelial, zebrafish

Integration of vascular progenitors into functional blood vessels represents a novel mechanism of vascular growth

“…To visualize SVF cells in live embryos, we utilized the CRISPR/Cas9-mediated nonhomologous integration approach (Auer et al, 2014) to knock-in a fast-folding Venus reporter into the endogenous etv2 locus and generated a stable etv2 Gt(2A-Venus)ci48 reporter line (further abbreviated as etv2 ) (Figure S3A). etv2 Gt(2A-Venus)+/embryos displayed Venus expression in vascular endothelial progenitors and differentiated vascular endothelial cells, red blood cells and macrophages, similar to the previously established Gal4 knock-in line etv2 ; UAS:GFP (Chestnut and Sumanas, 2019) (Figure S3B-G). While heterozygous etv2 Gt(2A-Venus)+/embryos appeared normal, homozygous etv2 Gt(2A-Venus)-/embryos showed severe defects in vascular development, associated with the etv2 loss-of-function phenotype.…”

Single-cell transcriptomic analysis identifies the conversion of zebrafish Etv2-deficient vascular progenitors into skeletal muscle

“…11e-h). In addition, GFP expression in skeletal muscle cells was also observed in etv2 y11−/− mutant embryos crossed into the previously established Tg(-2.3 etv2:GFP) reporter line but not in wild-type Tg(-2.3 etv2:GFP) 14,32 embryos (Fig. 3o).…”

“…Also, this population may have low-GFP expression as these cells may have just initiated etv2 expression, making them particularly difficult to detect by FACS. The etv2 ci32Gt ; UAS:GFP line is significantly brighter than the Tg(-2.3 etv2:GFP) line, likely due to the amplification of GFP expression by the Gal4:UAS system 14 . Therefore low GFP cells may have been missed when sorting cells from the Tg(-2.3 etv2:GFP) line.…”

“…1b, e). To confirm this result, we analyzed expression of RBC-specific markers using data from the recently reported global transcriptome study of etv2 ci32Gt heterozygous and homozygous embryos 14 . Indeed, expression of multiple RBCspecific genes, including hbaa1, vwf, hbae3 and others was significantly upregulated in etv2 ci32Gt homozygous embryos (Supplementary Table 2).…”

“…To analyze the diversity of hematovascular progenitors, we performed single-cell RNA-seq of GFP-positive cells sorted by fluorescence-activated cell sorting (FACS) from heterozygous and homozygous etv2 ci32Gt ; UAS:GFP zebrafish embryos at the 20somite stage. This reporter line, recently generated by CRISPR mediated homology-independent repair, has an insertion of the gal4 reporter within the etv2 coding sequence 14 . As described previously, heterozygous etv2 ci32Gt embryos recapitulate the endogenous expression pattern of etv2 in vascular endothelial progenitors and differentiated vascular endothelial cells, while homozygous embryos show profound defects in vascular development due to the interruption of the etv2 coding sequence 14 ( Supplementary Fig.…”